Autophagy‐related gene Atg5 is essential for astrocyte differentiation in the developing mouse cortex

Astrocyte differentiation is essential for late embryonic brain development, and autophagy is active during this process. However, it is unknown whether and how autophagy regulates astrocyte differentiation. Here, we show that Atg5, which is necessary for autophagosome formation, regulates astrocyte differentiation. Atg5 deficiency represses the generation of astrocytes in vitro and in vivo. Conversely, Atg5 overexpression increases the number of astrocytes substantially. We show that Atg5 activates the JAK2‐STAT3 pathway by degrading the inhibitory protein SOCS2. The astrocyte differentiation defect caused by Atg5 loss can be rescued by human Atg5 overexpression, STAT3 overexpression, and SOCS2 knockdown. Together, these data demonstrate that Atg5 regulates astrocyte differentiation, with potential implications for brain disorders with autophagy deficiency.     

Id4 is required for the correct timing of neural differentiation

Complex intrinsic and extrinsic mechanisms determine neural cell fate during development of the nervous system. Using Id4 deficient mice, we show that Id4 is required for normal development of the central nervous system (CNS), timing neural differentiation in the developing forebrain. In the absence of Id4, the ventricular zone of the neocortex, future hippocampus as well as lateral and medial ganglionic eminences exhibited a 20-30% reduction in mitotic neural precursor cells (NPCs). Although the number of apoptotic cells was significantly increased, the neocortex of Id4(-/-) embryos was consistently thicker due to premature neuronal differentiation, which resulted in an increase in early-born neurons in the adult Id4(-/-) cortex. Late-born cortical neurons and astrocytes in the cortex, septum, hippocampus and caudate putamen of Id4(-/-) adult brains were decreased, however, likely due to the depletion of the NPC pool. Consequently, adult Id4(-/-) brains were smaller and exhibited enlarged ventricles. In vitro analysis of neurosphere cultures revealed that proliferation of Id4-deficient NPCs was impaired and that BMP2-mediated astrocyte differentiation was accelerated in the absence of Id4. Together, these in vivo and in vitro data suggest a crucial role for Id4 in regulating NPC proliferation and differentiation.     

An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export

Gle1 is required for mRNA export in yeast and human cells. Here, we report that two human Gle1 (hGle1) isoforms are expressed in HeLa cells (hGle1A and B). The two encoded proteins are identical except for their COOH-terminal regions. hGle1A ends with a unique four-amino acid segment, whereas hGle1B has a COOH-terminal 43-amino acid span. Only hGle1B, the more abundant isoform, localizes to the nuclear envelope (NE) and pore complex. To test whether hGle1 is a dynamic shuttling transport factor, we microinjected HeLa cells with recombinant hGle1 and conducted photobleaching studies of live HeLa cells expressing EGFP-hGle1. Both strategies show that hGle1 shuttles between the nucleus and cytoplasm. An internal 39-amino acid domain is necessary and sufficient for mediating nucleocytoplasmic transport. Using a cell-permeable peptide strategy, we document a role for hGle1 shuttling in mRNA export. An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA. Coincidentally, SD peptide-treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1. These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.     

PKC-dependent ERK phosphorylation is essential for P2X7 receptor-mediated neuronal differentiation of neural progenitor cells

Purinergic receptors have been shown to be involved in neuronal development, but the functions of specific subtypes of P2 receptors during neuronal development remain elusive. In this study we investigate the distribution of P2X₇ receptors (P2X₇Rs) in the embryonic rat brain using in situ hybridization. At E15.5, P2X₇R mRNA was observed in the ventricular zone and subventricular zone, and colocalized with nestin, indicating that P2X₇R might be expressed in neural progenitor cells (NPCs). P2X₇R mRNA was also detected in the subgranular zone and dentate gyrus of the E18.5 and P4 brain. To investigate the roles of P2X₇R and elucidate its mechanism, we established NPC cultures from the E15.5 rat brain. Stimulation of P2X₇Rs induced Ca²⁺ influx, inhibited proliferation, altered cell cycle progression and enhanced the expression of neuronal markers, such as TUJ1 and MAP2. Similarly, knockdown of P2X₇R by shRNA nearly abolished the agonist-stimulated increases in intracellular Ca²⁺ concentration and the expression of TUJ1 and NeuN. Furthermore, stimulation of P2X₇R induced activation of ERK1/2, which was inhibited by the removal of extracellular Ca²⁺ and treatment with blockers for P2X₇R and PKC activity. Stimulation of P2X₇R also induced translocation of PKCα and PKCγ, but not of PKCβ, whereas knockdown of either PKCα or PKCγ inhibited ERK1/2 activation. Inhibition of PKC or p-ERK1/2 also caused a decrease in the number of TUJ1-positive cells and a concomitant increase in the number of GFAP-positive cells. Taken together, the activation of P2X₇R in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway.     

Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).     

Sema4D/PlexinB1 promotes endothelial differentiation of dental pulp stem cells via activation of AKT and ERK1/2 signaling

Inducing of dental pulp stem cells (DPSCs) into endothelial cells (ECs) to prevascularize pulp tissue constructs may offer a novel and viable approach for enhancing pulp regeneration. However, there are numerous challenges in current methods for the acquisition of sufficient translational ECs. It was known that Sema4D/PlexinB1 signaling exerts profound effects on enhancing vascular endothelial growth factor (VEGF) secretion and angiogenesis. Whether Sema4D/PlexinB1 could regulate endothelial differentiation of DPSCs is not yet investigated. In this study, when DPSCs were treated with Sema4D (2 μg/mL), ECs-specific (VEGFR1, VEGFR2, CD31, and vWF), and angiogenic genes and proteins were significantly upregulated. The induced ECs exhibited similar endothelial vessel formation ability to that of human umbilical vein endothelial cells (HUVECs). Furthermore, phosphorylation of AKT increased dramatically within 5 minutes (from 0.93 to 21.8), while p-ERK1/2 was moderately elevated (from 0.94 to 2.65). In summary, our results demonstrated that Sema4D/PlexinB1 signaling induces endothelial differentiation of DPSCs. The interactions of Sema4D, VEGF, ANGPTL4, ANG1, and HIF-1α may play a crucial role in mediating the differentiation process.     

OLIG2 Drives Abnormal Neurodevelopmental Phenotypes in Human iPSC-Based Organoid and Chimeric Mouse Models of Down Syndrome

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Immediate expression of Cdh2 is essential for efficient neural differentiation of mouse induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) exhibit reduced efficiency and higher variability in neural differentiation compared to embryonic stem cells (ESCs). In this study, we showed that mouse iPSCs failed to efficiently give rise to neuronal cells using conventional methods previously established for driving mouse ESC differentiation. We reported a novel approach which remarkably increases neural differentiation of mouse iPSCs. This novel approach initiated embryoid body (EB) formation directly from the whole cell clones isolated from the top of feeder cells. Compared to conventional neural induction methods such as single cell suspension or monolayer culture, the cell clone-derived EB method led to a pronounced increase in directed generation of various types of neural cells including neural stem cells, motoneurons and dopaminergic neurons in response to different inducers. Through gene expression microarray analysis, we identified 14 genes that were highly expressed in the cell clone-derived EBs. Among them, we found that Cdh2, also known as N-cadherin, played important roles in controlling the neural differentiation efficiency of mouse iPSCs. Forced expression of Cdh2 in iPSCs substantially enhanced the differentiation efficiency while knocking-down of Cdh2 by shRNA blocked the neural differentiation. Our results revealed a critical role of Cdh2 in the process of efficient neural differentiation of mouse iPS cells.     

SUMOylation mediates the disassembly of the Smad4 nuclear export complex via RanGAP1 in KELOIDS

Sentrin/small ubiquitin-like modifier (SUMO) has emerged as a powerful mediator regulating biological processes and participating in pathophysiological processes that cause human diseases, such as cancer, myocardial fibrosis and neurological disorders. Sumoylation has been shown to play a positive regulatory role in keloids. However, the sumoylation mechanism in keloids remains understudied. We proposed that sumoylation regulates keloids via a complex. RanGAP1 acted as a synergistic, functional partner of SUMOs in keloids. Nuclear accumulation of Smad4, a TGF-β/Smad pathway member, was associated with RanGAP1 after SUMO1 inhibition. RanGAP1*SUMO1 mediated the nuclear accumulation of Smad4 due to its impact on nuclear export and reduction in the dissociation of Smad4 and CRM1. We clarified a novel mechanism of positive regulation of sumoylation in keloids and demonstrated the function of sumoylation in Smad4 nuclear export. The NPC-associated RanGAP1*SUMO1 complex functions as a disassembly machine for the export receptor CRM1 and Smad4. Our research provides new perspectives for the mechanisms of keloids and nucleocytoplasmic transport.     

Calcineurin initiates smooth muscle differentiation in neural crest stem cells

The process of vascular smooth muscle cell (vSMC) differentiation is critical to embryonic angiogenesis. However, despite its importance, the vSMC differentiation program remains largely undefined. Murine gene disruption studies have identified several gene products that are necessary for vSMC differentiation, but these methodologies cannot establish whether or not a factor is sufficient to initiate the differentiation program. A gain-of-function system consisting of normal vSMC progenitor cells would serve as a useful complement to whole animal loss-of-function studies. We use such a system here, namely freshly isolated rat neural crest stem cells (NCSCs), to show that activation of the calcineurin signaling pathway is sufficient to drive these cells toward a smooth muscle fate. In addition, we present data suggesting that transforming growth factor (TGF)-beta1, which also causes NCSCs to differentiate into smooth muscle, activates calcineurin signaling in NCSCs, leading to a model in which activation of calcineurin signaling is the mechanism by which TGF-beta1 causes SMC differentiation in these cells.     

Tsukushi is essential for proper maintenance and terminal differentiation of mouse hippocampal neural stem cells

Secreted proteoglycan molecule Tsukushi (TSK) regulates various developmental processes, such as early body patterning and neural plate formation by interacting with major signaling pathways like Wnt, BMP, Notch etc. In central nervous system, TSK inhibits Wnt signaling to control chick retinal development. It also plays important roles for axon guidance and anterior commissure formation in mouse brain. In the present study, we investigated the role of TSK for the development and proper functioning of mouse hippocampus. We found that TSK expression is prominent at hippocampal regions of early postnatal mouse until postnatal day 15 and gradually declines at later stages. Hippocampal dimensions are affected in TSK knockout mice (TSK-KO) as shown by reduced size of hippocampus and dentate gyrus (DG). Interestingly, neural stem cell (NSC) density at the neural niche of DG was higher in TSK-KO compared with wild-type. The ratio of proliferating NSCs as well as the rate of overall cell proliferation was also higher in TSK-KO hippocampus. Our in vitro study also suggests an increased number of neural stem/progenitor cells residing in TSK-KO hippocampus. Finally, we found that the terminal differentiation of NSCs in TSK-KO was disturbed as the differentiation to neuronal cell lineage was increased while the percentages of astrocytes and oligodendrocytes were decreased. Overall, our study establishes the involvement of TSK in hippocampal development, NSC maintenance and terminal differentiation at perinatal stages.     

Three‐dimensional neural differentiation of embryonic stem cells with ACM induction in microfibrous matrices in bioreactors

The clinical use of pluripotent stem cell (PSC)‐derived neural cells requires an efficient differentiation process for mass production in a bioreactor. Toward this goal, neural differentiation of murine embryonic stem cells (ESCs) in three‐dimensional (3D) polyethylene terephthalate microfibrous matrices was investigated in this study. To streamline the process and provide a platform for process integration, the neural differentiation of ESCs was induced with astrocyte‐conditioned medium without the formation of embryoid bodies, starting from undifferentiated ESC aggregates expanded in a suspension bioreactor. The 3D neural differentiation was able to generate a complex neural network in the matrices. When compared to 2D differentiation, 3D differentiation in microfibrous matrices resulted in a higher percentage of nestin‐positive cells (68% vs. 54%) and upregulated gene expressions of nestin, Nurr1, and tyrosine hydroxylase. High purity of neural differentiation in 3D microfibrous matrix was also demonstrated in a spinner bioreactor with 74% nestin + cells. This study demonstrated the feasibility of a scalable process based on 3D differentiation in microfibrous matrices for the production of ESC‐derived neural cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1013–1022, 2013     

神经干细胞体外增殖分化的钙成像研究

神经干细胞具有广阔的应用前景,但对于其增殖和分化的内源机制、外部环境信号还并不十分了解。研究表明,钙信号很可能在其中起到了调控作用。利用钙离子成像技术,观察神经干细胞的单细胞体外增殖和分化过程,记录了在细胞分裂过程中钙信号变化的曲线。发现细胞增殖和分化过程中都会产生钙浓度的变化,但在细胞分裂后期两者钙信号的模式却存在差别。实验结果提示,胞内钙水平的波动只是细胞增殖的伴随产物,但却是细胞分化的必要条件。由此提出钙信号对神经干细胞分化调控机制的假设,并指出其对今后研究的意义。     

MFN2 silencing promotes neural differentiation of embryonic stem cells via the Akt signaling pathway

Mitofusin 2 (MFN2) is a regulatory protein participating in mitochondria dynamics, cell proliferation, death, differentiation, and so on. This study aims at revealing the functional role of MFN2 in the pluripotency maintenance and primitive differetiation of embryonic stem cell (ESCs). A dox inducible silencing and routine overexpressing approach was used to downregulate and upregulate MFN2 expression, respectively. We have compared the morphology, cell proliferation, and expression level of pluripotent genes in various groups. We also used directed differentiation methods to test the differentiation capacity of various groups. The Akt signaling pathway was explored by the western blot assay. MFN2 upregulation in ESCs exhibited a typical cell morphology and similar cell proliferation, but decreased pluripotent gene markers. In addition, MFN2 overexpression inhibited ESCs differentiation into the mesendoderm, while MFN2 silencing ESCs exhibited a normal cell morphology, slower cell proliferation and elevated pluripotency markers. For differentiation, MFN2 silencing ESCs exhibited enhanced three germs' differentiation ability. Moreover, the protein levels of phosphorylated Akt308 and Akt473 decreased in MFN2 silenced ESCs, and recovered in the neural differentiation process. When treated with the Akt inhibitor, the neural differentiation capacity of the MFN2 silenced ESCs can reverse to a normal level. Taken together, the data indicated that the appropriate level of MFN2 expression is essential for pluripotency and differentiation capacity in ESCs. The increased neural differentiation ability by MFN2 silencing is strongly related to the Akt signaling pathway.     

Activation of JNKs is essential for BMP9-induced osteogenic differentiation of mesenchymal stem cells

Although BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cell (MSCs), the molecular mechanism involved remains to be fully elucidated. Here, we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 stimulated the activation of JNKs in MSCs. BMP9-induced osteogenic differentiation of MSCs was dramatically inhibited by JNKs inhibitor SP600125. Moreover, BMP9-activated Smads signaling was decreased by SP600125 treatment in MSCs. The effects of inhibitor are reproduced with adenoviruses expressing siRNA targeted JNKs. Taken together, our results revealed that JNKs was activated in BMP9-induced osteogenic differentiation of MSCs. What is most noteworthy, however, is that inhibition of JNKs activity resulted in reduction of BMP9-induced osteogenic differentiation of MSCs, implying that activation of JNKs is essential for BMP9 osteoinductive activity. [BMB Reports 2013; 46(8):422-427]